A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/)

Global Carbon Budget 2023

Accurate assessment of anthropogenic carbon dioxide (CO2) emissions and their redistribution among the atmosphere, ocean, and terrestrial biosphere in a changing climate is critical to better understand the global carbon cycle, support the development of climate policies, and project future climate change. Here we describe and synthesize data sets and methodology to quantify the five major components of the global carbon budget and their uncertainties. Fossil CO2 emissions (EFOS) are based on energy statistics and cement production data, while emissions from land-use change (ELUC), mainly deforestation, are based on land-use and land-use change data and bookkeeping models. Atmospheric CO2 concentration is measured directly, and its growth rate (GATM) is computed from the annual changes in concentration. The ocean CO2 sink (SOCEAN) is estimated with global ocean biogeochemistry models and observation-based fCO2 products. The terrestrial CO2 sink (SLAND) is estimated with dynamic global vegetation models. Additional lines of evidence on land and ocean sinks are provided by atmospheric inversions, atmospheric oxygen measurements, and Earth system models. The resulting carbon budget imbalance (BIM), the difference between the estimated total emissions and the estimated changes in the atmosphere, ocean, and terrestrial biosphere, is a measure of imperfect data and incomplete understanding of the contemporary carbon cycle. All uncertainties are reported as ±1σ. For the year 2022, EFOS increased by 0.9 % relative to 2021, with fossil emissions at 9.9±0.5 Gt C yr−1 (10.2±0.5 Gt C yr−1 when the cement carbonation sink is not included), and ELUC was 1.2±0.7 Gt C yr−1, for a total anthropogenic CO2 emission (including the cement carbonation sink) of 11.1±0.8 Gt C yr−1 (40.7±3.2 Gt CO2 yr−1). Also, for 2022, GATM was 4.6±0.2 Gt C yr−1 (2.18±0.1 ppm yr−1; ppm denotes parts per million), SOCEAN was 2.8±0.4 Gt C yr−1, and SLAND was 3.8±0.8 Gt C yr−1, with a BIM of −0.1 Gt C yr−1 (i.e. total estimated sources marginally too low or sinks marginally too high). The global atmospheric CO2 concentration averaged over 2022 reached 417.1±0.1 ppm. Preliminary data for 2023 suggest an increase in EFOS relative to 2022 of +1.1 % (0.0 % to 2.1 %) globally and atmospheric CO2 concentration reaching 419.3 ppm, 51 % above the pre-industrial level (around 278 ppm in 1750). Overall, the mean of and trend in the components of the global carbon budget are consistently estimated over the period 1959–2022, with a near-zero overall budget imbalance, although discrepancies of up to around 1 Gt C yr−1 persist for the representation of annual to semi-decadal variability in CO2 fluxes. Comparison of estimates from multiple approaches and observations shows the following: (1) a persistent large uncertainty in the estimate of land-use changes emissions, (2) a low agreement between the different methods on the magnitude of the land CO2 flux in the northern extra-tropics, and (3) a discrepancy between the different methods on the strength of the ocean sink over the last decade

Climate change projections using the IPSL-CM5 Earth System Model: from CMIP3 to CMIP5

We present the global general circulation model IPSL-CM5 developed to study the long-term response of the climate system to natural and anthropogenic forcings as part of the 5th Phase of the Coupled Model Intercomparison Project (CMIP5). This model includes an interactive carbon cycle, a representation of tropospheric and stratospheric chemistry, and a comprehensive representation of aerosols. As it represents the principal dynamical, physical, and bio-geochemical processes relevant to the climate system, it may be referred to as an Earth System Model. However, the IPSL-CM5 model may be used in a multitude of configurations associated with different boundary conditions and with a range of complexities in terms of processes and interactions. This paper presents an overview of the different model components and explains how they were coupled and used to simulate historical climate changes over the past 150 years and different scenarios of future climate change. A single version of the IPSL-CM5 model (IPSL-CM5A-LR) was used to provide climate projections associated with different socio-economic scenarios, including the different Representative Concentration Pathways considered by CMIP5 and several scenarios from the Special Report on Emission Scenarios considered by CMIP3. Results suggest that the magnitude of global warming projections primarily depends on the socio-economic scenario considered, that there is potential for an aggressive mitigation policy to limit global warming to about two degrees, and that the behavior of some components of the climate system such as the Arctic sea ice and the Atlantic Meridional Overturning Circulation may change drastically by the end of the twenty-first century in the case of a no climate policy scenario. Although the magnitude of regional temperature and precipitation changes depends fairly linearly on the magnitude of the projected global warming (and thus on the scenario considered), the geographical pattern of these changes is strikingly similar for the different scenarios. The representation of atmospheric physical processes in the model is shown to strongly influence the simulated climate variability and both the magnitude and pattern of the projected climate changes

The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward λ▿

Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxBleft and boxBright reporters. Single amino acid substitutions complementing P22 N− virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxBleft and boxBright, suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, λ, and φ21 N proteins produced mutants that displayed bias. P22 N− plaque size plotted against boxBleft and boxBright reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both λ N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and φ21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and λ N−. The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition